We have used Drosophila models of SCA1 and HD for a comparative analysis of genetic factors involved in Ataxin-1 and Huntingtin-induced neurotoxicity. For that we have used, mainly, previously identified genetic modifiers of the SCA1 model.
We found genetic modifiers involved in a variety of cellular functions that affect similarly the SCA1 and HD Drosophila models. For other genes we did not detect modification of Huntingtin toxicity. Surprisingly, we also identified modifier genes that have antagonistic effects on Ataxin-1 and Huntingtin toxicity. Some of the opposing interesting modifiers were validated in the CNS using a neuronal assay, such as the climbing assay. We have also found no consistent correlation between modifier gene activity and nuclear inclusion formation.
To our knowledge, the study here presented represents the largest study to date comparing genetic modifiers between two polyglutamine disorders using fly models. However, and as most of the genetic screens preformed, it is hard to identify a clear link between the observation of a genetic modifier that modulates specific phenotypes characteristic of the model being studied and a mechanism that explains the toxicity observed. Because of that, many have considered that genetic screens are merely a large collection of observations. However, we believe that the fact that two disorders have been compared in this study overcomes this negative aspect of the approach we have taken, in the sense that it may contribute to understanding why, at least for some specific cases, some of the characteristic clinical and pathological phenotypes polyglutamine diseases are so distinctive.
Many of the genes highlighted in this study can be the staring point for us or other groups to start a line of research on a specific gene, interaction or effect. However, our results support a model in which each disease should be analyzed separately and not in the context of other diseases. As we have shown, a genetic modifier for one of the diseases might have the opposite
effect in another one. Therefore, therapeutic approaches need to be carefully directed and taken into consideration the specific protein causing the disease (and its protein context) and not what is common among that specific group of diseases.
The results here present will also contribute to the creation of a network of proteins that interact with the disease-causing proteins. This will allow a better assessment of all the mechanisms and pathways leading to the toxicity observed not only in the animal models, but also, ultimately, in the patients. The work here presented does not intend to establish specific mechanisms or pathways leading to toxicity in polyglutamine disorders, but yet aims to contribute to the knowledge of the network of interactions behind the mechanisms that cause the disease clinical, phenotypical and molecular phenotypes observed in patients.
Furthermore, this work reinforces the importance of using genetic approaches in order to fully understand mechanisms underlying disease pathology, and also in this case, the relevance of the protein context for the development or progression of polyglutamine diseases and for the occurrence of distinct characteristic diseases phenotypes among this group of disorders.
Our results also support the recent evidences that focus on the function of the mutant protein itself and how protein context (and not just the polyglutamine tract on its own) can regulate the pathogenic events in each of these diseases.
As mentioned, for this study we have used genes previously identified as genetic modifiers of the SCA1 model developed in the Botas laboratory (Fernandez-Funez et al., 2000). As a result of this approach, many modifiers might have not been identified, and, therefore, an interesting approach would be to test some know HD model genetic modifiers (for example some of the genes identified in the study by Hughes and colleagues (Kaltenbach et al., 2007)) in the SCA1 fly model developed in the Botas Laboratory. Also interesting would be to test some of the known- SCA1 genetic modifiers in the newly developed full length HD model (Eliana Romero and Juan Botas,
submitted). He know now that a collection of various proteins and pathways are involved in polyglutamine-induced neurodegeneration and that, most likely, some will be more relevant for one disorder than to another. The combination of the studies mentioned here and our study will likely open new and interesting avenues of research.
Figure 58 - Model for Cellular Pathogenesis. The figure here presented was specifically designed to illustrate some of the possible mechanism involved in cellular pathogenesis in HD. With the obvious exception that Huntingtin is predominantly a cytoplasmic protein, many of the mechanism here schematically represented can be adjusted to other polyglutamine disorders. For the specific case of HD, the normal function of Htt is yet to be clarified, but it is possible that it is involved in cytoskeletal functions or vesicle recycling. It may cycle to the nucleus and have a normal role in the regulation of gene transcription, but more studies will be needed in order to confirm this observation. The conformational changes resulting from the expansion of the trinucleotide repeats lead to unfolding/abnormal folding of the protein, and thus molecular chaperones can play an important role in this process. Proteolitic cleavage of the mutant protein can also occur, both in the cytoplasm and in the nucleus, depending on the protein. The mutant protein, once in the nucleus, forms NIs (note that in some cases NIs in the cytoplasm can also be observed). As discussed thought this thesis, the role for toxicity of these aggregates of mutant protein and additional proteins (trapped in these aggregates) is still matter of much debate. However, nuclear toxicity is believed to be caused by disturbance of gene transcription, one of the main common molecular features of
One other line of investigation not mentioned in this thesis that is gaining relevance in the field is miRNAs. miRNAs have been implicated in many human diseases (reviewed in Hebert and De Strooper, 2007), and in particular in neurodegenerative diseases (Bilen et al., 2006a; Bilen et al., 2006b; Kim et al., 2007). Therefore, their role in the pathogenic mechanism is anticipated to be of greater importance than previously thought. However, more work will be needed in order to understand the clinical relevance of these molecules and, specially, to clearly define molecularly how they influence pathogenesis. Not only the miRNAs need to be the focus of more detailed studies, but also their interacting proteins and enzymes need to be further investigated.
Finding the mechanism by which the polyglutamine tract, and more interestingly, how the protein context of one specific protein, affects the activity of the disease protein as a whole might help research aimed at developing a therapeutic solution of this type of disorders. Either by interfering with the specific interactors that mediate the selective neuronal toxicity or by modulating the downstream events of those interactions, it will be possible to develop a therapeutic approach in the future. The findings obtained as a result of this PhD thesis reinforce that, in order to achieve effective treatments, each disease needs to be carefully studied in its own context, and not as part of a group of disorders.