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Glassy eyes: A new transgenic Drosophila model for exploring human prion protein toxicity

Posted by , on 27 April 2022

This post highlights the approach and finding of a new research article published by Disease Models and Mechanisms (DMM). This feature is written by Melanie Mew as part of a seminar at The University of Alabama (taught by DMM Editorial Board member, Professor Guy Caldwell) on current topics related to use of animal and cellular model systems in the studies of human disease.

Prions are misfolded proteins that can induce other proteins to fold abnormally and are transmissible from one individual to another. Prion diseases (PrDs) range from sporadic forms of bovine spongiform encephalopathy (“mad cow” disease) to genetically inherited Creutzfeldt-Jacob disease (CJD). They are uniformly fatal and untreatable. The key to preventing and treating PrDs may lie in understanding the structure of prions and how they mediate neurodegeneration. A major challenge to studying PrDs is that they occur only in mammals, whose long generation times and high maintenance costs limit the speed and statistical power of research. Smaller models such as Drosophila are a useful alternative because evolutionary conservation of basic cellular processes allows them to recapitulate PrD phenotypes. A remaining mystery in PrD research is the mechanisms through which prions cause disease. In a recent study published in Disease Models and Mechanisms, Myers et al., (2022) use a transgenic Drosophila model expressing human or rodent prion protein (PrP) to elucidate intrinsic and extrinsic mediators of prion toxicity. The researchers identified a new “glassy eyes” phenotype, associated with reduced size and organization of the eyes, in flies expressing toxic forms of PrP.

An initial investigation of human PrP compared to the less toxic rodent PrP found that, while these proteins share many similarities, amino acid changes in the C-terminal 3D (CT3D) may mediate the greater instability and toxicity of human PrP. Expressing hamster PrP in fruit flies permits normal eye development (Fernandez-Funez et al., 2017), whereas random insertion of human PrP into the Drosophila genome is deleterious to eye development (Myers et al., 2022). Specifically, comparison of two naturally occurring human PrP polymorphisms: Met129 and Val129—the latter associated with  associated with a higher probability of developing CJD—induced a glassy-eyed phenotype. In separate experiments, a conditional activator system was used to induce pan-neuronal hamster or human PrP expression in adult flies. These individuals were subjected to a climbing assay, in which the human PrP flies developed locomotor deficits significantly worse than those of hamster PrP-expressing flies. Human PrP expression was also associated with reduced size and structure of Drosophila mushroom bodies, which are non-essential features responsible for learning and memory in flies and other insects. 

To directly compare the toxicity of rodent and human PrP, Myers et al. inserted codon-optimized versions of each gene at a previously validated attP2 locus (Bischof et al., 2007, Moore et al., 2018). They created flies expressing either mouse PrP, human PrP-M129, or human PrP-V129. Flies expressing mouse PrP had eyes comparable to those of controls, whereas flies homozygous for either form of human PrP displayed glassy eyes. Having established a clear pattern of toxicity associated with human PrP, the researchers next investigated genetic and molecular mediators of human PrP toxicity. Specific extrinsic mediators of PrP toxicity may include members of the unfolded protein response (UPR) pathway, associated with ER stress. The UPR is activated by various sensors, including Ire1α and PERK. Ire1α and a protein activated downstream of it, X-box binding protein 1 (XBP1), can protect against human PrP toxicity. Silencing either of these proteins alone has no effect on eye development, but produces a small-eye phenotype when combined with human PrP expression. Conversely, silencing PERK or its downstream affiliate, ATF4, independently or in combination with human PrP expression allows for normal eye development. Therefore, activating the PERK branch of the UPR or inhibiting the Ire1α branch could be valuable treatment strategies for PrDs. Other therapeutic options for PrDs could include modulating the structure of PrP itself. In this vein, Myers and colleagues investigated the protective effects of amino acid substitutions found in dogs, horses, rabbits, and pigs. Asp167Ser, the canine residue mutation, was protective against eye disorganization and mushroom body degeneration in flies.

The research performed by Myers and colleagues suggests that human PrDs may be treatable with drugs that stabilize the CT3D of PrP or modulate the UPR response to PrP. Future research will likely further elucidate the interacting partners of human PrP that mediate neurodegeneration, as well as the roles of specific residues in contributing to intrinsic protein stability. Understanding the evolution of human PrP could also help identify benefits of stability-sacrificing human PrP mutations. Microbial prions, for example, have been shown to sometimes confer evolutionary adaptations such as stress tolerance and memory (Levkovich et al., 2021). For now, this work informs our general understanding of neurodegeneration induced by protein aggregation—a hallmark of other disorders such as Parkinson’s disease and Alzheimer’s disease (Soto and Pritzkow, 2018)—and is a step toward discovering effective treatments for neurological diseases.

REFERENCES

Bischof J., Maeda R.K., Hediger M., Karch F. and Basler K. (2007) An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proc Natl Acad Sci U S A. 104, 3312-7.

Fernandez-Funez P., Sanchez-Garcia J. and Rincon-Limas D.E. (2017) Drosophila models of prionopathies: insight into prion protein function, transmission, and neurotoxicity. Curr. Opin. Genet. Dev. 44, 141-148.

Levkovich S.A., Rencus-Lazar S., Gazit E., Laor Bar-Yosef D. (2021) Microbial Prions: Dawn of a New Era. Trends Biochem. Sci. 46, 391-405.

Moore, B. D., Martin, J., de Mena, L., Sanchez, J., Cruz, P. E., Ceballos-Diaz, C., Ladd, T. B., Ran, Y., Levites, Y., Kukar, T. L., Kurian, J. J., McKenna, R., Koo, E. H., Borchelt, D. R., Janus, C., Rincon-Limas, D., Fernandez-Funez, P. and Golde, T. E. (2018). Short Aβ peptides attenuate Aβ42 toxicity in vivo. J. Exp. Med. 215, 283-301.

Myers, R., Sanchez-Garcia, J., Leving, D., Melvin, R. and Fernandez-Funez, P. (2022) New Drosophila models to uncover the intrinsic and extrinsic factors mediating the toxicity of the human prion protein. Dis. Model. Mech.

Soto, C. and Pritzkow, S. (2018) Protein misfolding, aggregation, and conformational strains in neurodegenerative diseases. Nat. Neurosci. 21, 1332–1340.

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