Federico Herrera

Tau enhances α-synuclein aggregation and toxicity in cellular models of synucleinopathy.

Background The simultaneous accumulation of different misfolded proteins in the central nervous system is a common feature in many neurodegenerative diseases. In most cases, co-occurrence of abnormal deposited proteins is observed in different brain regions and cell populations, but, in some instances, the proteins can be found in the same cellular aggregates. Co-occurrence of tau and α-synuclein (α-syn) aggregates has been described in neurodegenerative disorders with primary deposition of α-syn, such as Parkinsons disease and dementia with Lewy bodies. Although it is known that tau and α-syn have pathological synergistic effects on their mutual fibrillization, the underlying biological effects remain unclear. Methodology/Principal Findings We used different cell models of synucleinopathy to investigate the effects of tau on α-syn aggregation. Using confocal microscopy and FRET–based techniques we observed that tau colocalized and interacted with α-syn aggregates. We also found that tau overexpression changed the pattern of α-syn aggregation, reducing the size and increasing the number of aggregates. This shift was accompanied by an increase in the levels of insoluble α-syn. Furthermore, co-transfection of tau increased secreted α-syn and cytotoxicity. Conclusions/Significance Our data suggest that tau enhances α-syn aggregation and toxicity and disrupts α-syn inclusion formation. This pathological synergistic effect between tau and α-syn may amplify the deleterious process and spread the damage in neurodegenerative diseases that show co-occurrence of both pathologies.

Visualization of cell-to-cell transmission of mutant huntingtin oligomers.

We developed a new cell model for the visualization of toxic huntingtin oligomers in living cells. Huntingtin exon 1 (25Q or 103Q) was fused to non-fluorescent halves of the Venus protein. When huntingtin dimerizes inside the cells, Venus becomes functionally reconstituted and emits fluorescence. Oligomerization, aggregation and toxicity of mutant huntingtin were assessed by several procedures. We also present evidence that the transmission of huntingtin between cells can be determined in a quantitative manner with our model. Thus, this model can be a powerful screening tool for the identification of modifiers of oligomerization and cell-to-cell traffic of mutant huntingtin.

DJ-1 modulates aggregation and pathogenesis in models of Huntington's disease

The oxidation-sensitive chaperone protein DJ-1 has been implicated in several human disorders including cancer and neurodegenerative diseases. During neurodegeneration associated with protein misfolding, such as that observed in Alzheimers disease and Huntingtons disease (HD), both oxidative stress and protein chaperones have been shown to modulate disease pathways. Therefore, we set out to investigate whether DJ-1 plays a role in HD. We found that DJ-1 expression and its oxidation state are abnormally increased in the human HD brain, as well as in mouse and cell models of HD. Furthermore, overexpression of DJ-1 conferred protection in vivo against neurodegeneration in yeast and Drosophila. Importantly, the DJ-1 protein directly interacted with an expanded fragment of huntingtin Exon 1 (httEx1) in test tube experiments and in cell models and accelerated polyglutamine aggregation and toxicity in an oxidation-sensitive manner. Our findings clearly establish DJ-1 as a potential therapeutic target for HD and provide the basis for further studies into the role of DJ-1 in protein misfolding diseases.

α-Synuclein modifies huntingtin aggregation in living cells

Htt and alpha‐syn physically interact by comigration in non‐denaturing gel electrophoresis (View interaction)

α-Synuclein modifies mutant huntingtin aggregation and neurotoxicity in Drosophila

Protein misfolding and aggregation is a major hallmark of neurodegenerative disorders such as Alzheimers disease (AD), Parkinsons disease (PD) and Huntingtons disease (HD). Until recently, the consensus was that each aggregation-prone protein was characteristic of each disorder [α-synuclein (α-syn)/PD, mutant huntingtin (Htt)/HD, Tau and amyloid beta peptide/AD]. However, growing evidence indicates that aggregation-prone proteins can actually co-aggregate and modify each others behavior and toxicity, suggesting that this process may also contribute to the overlap in clinical symptoms across different diseases. Here, we show that α-syn and mutant Htt co-aggregate in vivo when co-expressed in Drosophila and produce a synergistic age-dependent increase in neurotoxicity associated to a decline in motor function and life span. Altogether, our results suggest that the co-existence of α-syn and Htt in the same neuronal cells worsens aggregation-related neuropathologies and accelerates disease progression.

Glycation potentiates neurodegeneration in models of Huntington's disease.

Protein glycation is an age-dependent posttranslational modification associated with several neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. By modifying amino-groups, glycation interferes with folding of proteins, increasing their aggregation potential. Here, we studied the effect of pharmacological and genetic manipulation of glycation on huntingtin (HTT), the causative protein in Huntington’s disease (HD). We observed that glycation increased the aggregation of mutant HTT exon 1 fragments associated with HD (HTT72Q and HTT103Q) in yeast and mammalian cell models. We found that glycation impairs HTT clearance thereby promoting its intracellular accumulation and aggregation. Interestingly, under these conditions autophagy increased and the levels of mutant HTT released to the culture medium decreased. Furthermore, increased glycation enhanced HTT toxicity in human cells and neurodegeneration in fruit flies, impairing eclosion and decreasing life span. Overall, our study provides evidence that glycation modulates HTT exon-1 aggregation and toxicity, and suggests it may constitute a novel target for therapeutic intervention in HD.

Imaging protein oligomerization in neurodegeneration using bimolecular fluorescence complementation.

Neurodegenerative disorders such as Alzheimers, Parkinsons, Huntingtons, or Prion diseases belong to a superfamily of pathologies known as protein misfolding disorders. The hallmark of these pathologies is the aberrant accumulation of specific proteins in beta sheet-rich amyloid aggregates either inside or outside cells. Current evidence suggests that oligomeric species, rather than mature protein aggregates, are the most toxic forms of the pathogenic proteins. This is due, at least in part, to their greater solubility and ability to diffuse between intracellular and extracellular compartments. Understanding how oligomerization occurs is essential for the development of new treatments for this group of diseases. Bimolecular fluorescence complementation assays (BiFC) have proved to be excellent systems to study aberrant protein-protein interactions, including those involved in neurodegenerative diseases. Here, we provide a detailed description of the rationale to develop and validate BiFC assays for the visualization of oligomeric species in living cells in the context of neurodegeneration. These systems could constitute powerful tools for the identification of genetic and pharmacological modifiers of protein misfolding and aggregation.

Studying the Molecular Determinants of Protein Oligomerization in Neurodegenerative Disorders by Bimolecular Fluorescence Complementation

The main feature of protein misfolding disorders is the presence of protein deposits in cells and tissues as a consequence of aberrant protein–protein interactions. Neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease belong to this class of disorders. The deposition of large amyloid deposits is a dynamic process that starts with the generation of small, soluble species known as dimers and oligomers. The identification of physical, chemical and genetic conditions that promote – or inhibit – the pathologic deposition of proteins is essential for understanding and preventing neurodegeneration. In this chapter, we explain the history, features, design, development and optimization of bimolecular fluorescence complementation (BiFC) assays for the study of protein oligomerization, aggregation and toxicity in neurodegenerative disorders. In BiFC assays protein chimeras containing the protein of interest fused to non-fluorescent halves of a fluorescent reporter protein are generated. When the proteins of interest interact, the halves of the fluorophore become close enough to reconstitute the fluorescence, which is therefore proportional to the production of dimers and oligomers of the proteins of interest. The fact that BiFC assays allow the direct visualization and measurement of the first steps of aggregation makes them a unique tool in the field of protein misfolding disorders. Additionally, BiFC assays require only basic cell and molecular biology materials and equipment, have a wide array of possible experimental settings/fluorophores, allow studies in living cells and organisms, and are especially suitable for large drug and genetic screenings.

B10 The role of n-terminal phosphorylation in huntingtin's oligomerisation, aggregation and toxicity

Background Aggregation of mutant huntingtin (Htt) is a dynamic process that starts with the association of a few misfolded Htt monomers in small, soluble oligomeric structures. Current evidence suggests that dimers and oligomers are the most toxic species and that larger aggregates are rather neuroprotective. In order to prevent Htt aggregation and toxicity it is essential to understand the molecular mechanisms of oligomerisation. Aims Elucidate the role of Htts N-terminal region in its oligomerisation, aggregation and toxicity, with particular emphasis on the phosphorylatable residues (T3, S13 and S16). Methods/techniques We have recently developed a cellular model for the visualisation of Htt oligomeric species in living cells, based on the bimolecular fluorescence complementation (BiFC) assay. In this model, Htt exon1 is fused to two non-fluorescent halves of the Venus protein (V1 and V2). When Htt dimerises, the two halves get together and reconstitute the functional fluorophore. We used this BiFC assay to create a series of phosphoresistant (T3A, S13A, S16A) and phosphomimic (T3D, S13D, S16D) Htt mutants. Results/outcome When phosphomimic mutations were present in both 103QHtt-V1 and 103QHtt-V2 BiFC constructs, the generation of inclusion bodies was completely abolished. However, the levels of oligomeric species were similar to the non-mutated and the phosphoresistant BiFC pairs. The combinations of a non-mutated construct with a phosphomimic construct produced intermediate phenotypes in terms of aggregation. Phosphoresistant BiFC pairs did not produce overt phenotypes. Mutations in N-terminal residues had varied effects on Htt toxicity, which apparently were not associated with the levels of oligomeric species or inclusion bodies. Conclusions The phosphorylation state of Htts N-terminal region is a key player in the formation of inclusion bodies and the toxicity of the protein.

A02 Visualisation of mutant huntingtin oligomerisation in living cells

Background Aggregation of mutant huntingtin is a dynamic process that starts with the association of a few misfolded huntingtin monomers in small, soluble oligomeric structures. Current evidence suggests that dimers and oligomers are the most toxic species of mutant huntingtin and that the largest aggregates are rather neuroprotective. In order to prevent huntingtin aggregation and toxicity it is essential to understand the molecular mechanisms of oligomerisation. However, existing experimental models of Huntingtons disease do not enable the direct visualisation of the smaller intermediary species in the aggregation process, only of the larger huntingtin aggregates. Aims To develop a cellular model for the visualisation and study of dimers and oligomers of mutant huntingtin in living cells. Methods We generated two different constructs that carried complementary portions of the Venus fluorescent reporter protein fused to the exon 1 of mutant huntingtin (103Q glutamine tract). When the exon-1 of mutant huntingtin dimerises inside the cells, both complementary halves of Venus reconstitute the functional fluorophore and emit green fluorescence, which can be easily visualised and measured. Results Oligomer generation and toxicity was evaluated over time and confirmed by different methods. We used similar fusion constructs with wild-type huntingtin exon 1 (25Q glutamine tract) as a negative control for oligomerisation and toxicity. Additional controls included, among others, the transfection of cells with only one of the constructs or with the two halves of the Venus protein without huntingtin. The robustness of our model is being tested by analysing the effect of known modifiers of aggregation, such as heat shock proteins, on huntingtin oligomerisation. Conclusions Our preliminary results indicate that this model can be a powerful tool for the identification of new molecular targets for the treatment of Huntingtons disease.