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The role of post-translational modifications on the oligomerization, aggregation and toxicity of mutant Huntingtin
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Neurodegenerative diseases (NDs) present major health and economic challenges to modern societies. No treatments can currently stop, delay or reverse the progression of such devastating disorders. The development of effective therapeutics is particularly complicated because the molecular underpinnings of NDs are still far from being understood. A possible clue could come from the fact that a large number of NDs display protein misfolding and aggregation as pathophysiological hallmarks. Understanding factors which modulate the aggregate formation process and the mechanisms by which protein intermediates exert cellular toxicity could advance our knowledge of disease pathogenesis and represents a possible intervention point in our search for effective therapies. Recent years have seen several rational targets for therapeutic development being generated from a combination of cell culture studies and analysis of genetically tractable animals. Here we used a cell model exploiting bimolecular fluorescence complementation (BiFC) and Drosophila models to study the molecular basis of Huntington’s disease (HD).
HD is a hereditary neurodegenerative disorder generally affecting individuals of 40-50 years of age, although the most severe forms of the disease can appear in children and adolescents. HD causes death invariably 15-20 years after the first symptoms, with patients and their families experiencing incalculable pain and suffering throughout the course of the disease. The causative mutation encodes a polyglutamine expansion in the huntingtin (HTT) protein, which modulates its aggregation and cytotoxicity by still unclear mechanisms. Although HD is genetically well defined, unidentified genetic and/or environmental factors may also play a role in disease development and progression.
The present thesis aimed to (i) characterize the effect of specific post-translational modifications (PTMs) upon mutant HTT aggregation dynamics and toxicity, as well as (ii) investigate the putative regulatory role of protein phosphatases in these processes, and (iii) understand how interactions between mutant HTT and other aggregation-prone proteins may modulate disease pathogenesis.
We started by determining the relative contribution of each of the three phosphorylatable residues (T3, S13 and S16) in the N-terminal region of HTT towards its aggregation and toxicity. By using single phosphomimic (D) and phosphoresistant (A) BiFC mutants, we found that all of the D mutants completely abolished the formation of large insoluble species, while having little effect on oligomerization and toxicity. When combined with non-phosphorylated forms of mutant HTT, D mutants differentially affected HTT aggregation dynamics in living cells, with T3 phosphorylation having the most dominant effect. Analyses of transgenic Drosophila mutants further supported T3 as a critical modulator of mutant HTT aggregation, with both larvae and adult fly tissues from T3D mutants exhibiting lower number of aggregates compared to non-mutated counterpart.
Since N-terminal phosphorylation strikingly prevented HTT aggregation, we investigated specific protein phosphatases as potential modulators of HTT aggregation and toxicity in both mammalian cells and Drosophila. An initial pharmacological screen in our BiFC cell model identified three phosphatase inhibitors (PP1, PP2A and Cdc25) as potential suppressors of HTT aggregation. Subsequent RNAi experiments in vivo showed that PP1 downregulation prevented the deposition of HTT inclusions in adult fly dopaminergic neurons, whereas both PP2 and Cdc25 knockdown did not produce overt phenotypes. Interestingly, downregulation of PP1 in flies caused severe age-dependent neurodegeneration. Together, these findings indicate that PP1 modulates HTT aggregation and toxicity in opposite ways, which further support the current notion that large inclusions are not the toxic factors in HD and other NDs.
The formation of advanced glycation-end products (AGEs) as result of the interaction between proteins and reducing sugars is referred to as protein glycation. Such irreversible PTM is found in various disease-associated proteins, and is thought to contribute to the pathogenesis of NDs. Here, we provide the first in vivo evidence on the deleterious impact of glycation on HTT biology and HD. Pharmacological or genetic modulation of protein glycation led to increased accumulation of AGEs in mutant HTT-expressing flies and exacerbated phenotypes related to both early and late stages of HD. In particular, we showed that treatment with increasing doses of a potent glycation agent - methylglyoxal (MGO) - accelerated neuronal loss in adult animals and negatively affected the development of photoreceptor neurons. Genetic suppression of the MGO pathway resulted in impaired development, reduced lifespan and increased neurodegeneration of HD flies. In humans, increased blood glucose levels activate the MGO pathway, which would implicate altered glucose metabolism (e.g. diabetes) as an environment risk factor for HD development.
Aberrant protein-protein interactions can compromise normal cell function and contribute to cytotoxicity in NDs. Here, we explored the effects of mutant HTT interactions with other aggregation-prone proteins on each other’s behavior. We showed that mutant HTT interacts and co-aggregates with alpha-synuclein (α-syn), Tau and the Rnq1 prion protein in living human cells, altering their subcellular localization and aggregation patterns. While in vivo α-syn and Tau studies were carried out by various collaborators, we set out to elucidate the interplay between the Rnq1 prion protein and mutant HTT. In yeast, the Q/N-rich Rnq1 prion protein is required for mutant HTT aggregation and toxicity. Because the human genome encodes multiple proteins enriched in Q/N domains, we hypothesized that they may function as modifiers of disease pathogenesis. Drosophila expressing Rnq1 showed a significant increase in age-dependent neuronal loss and motor dysfunction. Furthermore, as occurs in yeast, we found that Rnq1 potentiates mutant HTT toxicity in Drosophila. Together, our results are consistent with the existence of two mechanisms for HTT toxicity, one related to toxicity of the aggregation process itself, and the other related to the sequestration of molecules required for normal cell function.
Overall, our findings provide new insights into HD pathology by uncovering novel aspects of mutant HTT aggregation and toxicity. We expect these studies to contribute to a better understanding of the mechanisms underlying the pathogenesis of HD and other human protein misfolding disorders, and thus lay the groundwork for effective therapeutic strategies for these devastating disorders.
Descrição
Palavras-chave
Huntington’s disease Huntingtin Phosphorylation Glycation Prion proteins Teses de doutoramento - 2018