Supervisors info:
Ισιδώρα Σ. Παπασιδέρη
Καθηγήτρια, Τομέας Βιολογίας Κυττάρου και Βιοφυσικής,
Τμήμα Βιολογίας, ΕΚΠΑ
Δημήτριος Ι. Στραβοπόδης
Αναπληρωτής καθηγητής, Τομέας Βιολογίας Κυττάρου & Ανάπτυξης,
Τμήμα Βιολογίας, ΕΚΠΑ
Ευθύμιος Σκουλάκης
Αναπληρωτής Διευθυντής, Ερευνητής Α’, Τομέας Νευροεπιστημών
Ινστιτούτο Βασικής Βιοϊατρικής Έρευνας Ε.ΚΕ.ΒΕ «ΑΛΕΞΑΝΔΡΟΣ ΦΛΕΜΙΓΚ»
Summary:
During the past few years, the exponential increase in life expectancy has brought age-related diseases to the fore. Among others, Tauopathies such as Alzheimer's disease and frontotemporal dementia, which affect adults over 65 years of age, induce slow and gradual impairments in memory, judgment, comprehension and speech, impairment of daily functioning and spatiotemporal disorientation. Tauopathies, as nosological entities, are histopathologically characterized by intraneuronal accumulation of the hyperphosphorylated Tau protein, which changes its three-dimensional conformation and forms aggregates. This is by no means to be expected for a protein as water-soluble as Tau, whose sequence is natively disordered, stable in high temperatures and under acidic conditions, as well as rich in polar amino acids. However, under hitherto unexplained conditions, Tau loses its paperclip-like shaped conformation, which favors its binding to microtubules. Instead, Tau forms β-sheet structures, which interdigitate to form oligomers and in the final stage of Tauopathy mature aggregates.
The research team of Dr. Katerina Papanikolopoulou at the Biomedical Sciences Research Center "Alexander Fleming" attempted to elucidate the in vivo mechanism of Tau aggregation. For this reason, proteins were sought that interact with the human, wild-type Tau in the central nervous system of Drosophila melanogaster and that may be involved in specific intracellular signaling pathways. A series of proteomic experiments led to the unexpected identification of the Drosophila MICAL (Mical) as a cellular partner of Tau. Mical protein is a multidomain flavin monooxygenase, which oxidizes cysteine or methionine residues that are located on the surface of suitable substrates. In this way, Mical controls their action and at the same time becomes involved in brain developmental processes, such as dendritic pruning and axonal guidance or in pathological conditions that are related to actin-dependent processes. The effect of Mical on Tau toxicity was assessed by an eclosion assay. Results are presented as the ratio of flies co-expressing Tau-Mical or Tau-Mical RNAi over the number of flies expressing Tau alone. Interestingly, Mical elevation enhanced Tau toxicity (decreased eclosion) whereas Mical reduction attenuated Tau-dependent developmental lethality. Given the effects of Mical in human Tau-expressing flies, the laboratory decided to investigate the levels and localization of MICAL in samples (frozen tissue and paraffin sections) from non-demented patients as well as in patients with Tauopathies, namely Alzheimer’ disease and the distinctive form of Frontotemporal Dementia, Pick’s disease. In humans, there are three MICAL proteins (MICAL1, MICAL2 and MICAL3). Phylogenetic analysis of MICAL family members showed that Drosophila MICAL and Human MICAL1 are clustered in the same clade, hence the laboratoryinitially focused on this protein. A very exciting and novel result was that in the brain of Pick's disease patients, MICAL1 is significantly up-regulated compared to normal controls and co-localizes with Tau in the cytoplasmic inclusions known as Pick bodies. MICAL1 is also up-regulated in most of the AD samples tested but shows no immunoreactivity in paraffin sections.
The present thesis project was based on the genetic interactions between Mical and Tau with respect to a) Tau normal function as a cytoskeletal protein b) Tau isoform specific interactions c) Tau-associated neuronal toxic, as well as on the biochemical characterization of interaction between the two proteins. Six series of transgenic flies were created in a way that they could express a Tau transgene (0N3R, 0N4R, 1N3R, 1N4R, 2N3R, 2N4R) pan-neuronally using the GAL4/UAS system, by coexpression of a Mical transgene or by transgenic Mical RNAi respectively. These strains were used in simple western blot experiments or variants thereof, such as the aggregate isolation and microtubule binding protocol. Then, the toxicity of Mical-Tau interaction was tested in the adult Drosophila brain through longevity and resistance to oxidative stress assays and though observating the rough eye phenotype. Experiments on longevity and interaction study were based on Drosophila strains, where transgenic expression was monitored by the TARGET system.
In summary:
• Mical up-regulation increases all six Tau isoform levels in the CNS of Drosophila, while its down-regulation decreases their levels.
• Mical enhances the propensity aggregation of 2N3R and 2N4R Tau isoforms
• Mical does not affect the microtubule binding capacity of 2Ν3R and 2Ν4R, except from the case where 2N4R and MICAL-RNAi are co-expressed leading to the detachment of the 2Ν4R isoform from the microtubules
• Mical-Tau interaction exacerbates the 2N4R-mediated rough eye phenotype
• Mical overexpression or down-regulation seems to have no effect on Tau longevity curves, but this is due to a systematic error caused by incubator malfunction
• Mical overexpression increases the oxidative stress susceptibility of the flies expressing 2Ν3R or 2N4R pan-neuronally
• Mical down-regulation alters the interactome of Tau in the CNS of Drosophila
Keywords:
Mical, Tau isoforms, aggregates, microtubules, neurotoxicity
