New Drosophila models to uncover the intrinsic and extrinsic factors that mediate the toxicity of the human prion protein

Abstract

Misfolding of the prion protein (PrP) is responsible for devastating neurological disorders in humans and other mammals. An unresolved problem in the field is unraveling the mechanisms governing PrP conformational dynamics, misfolding, and the cellular mechanism leading to neurodegeneration. The variable susceptibility of mammals to prion diseases is a natural resource that can be exploited to understand the conformational dynamics of PrP. Here we present a new fly model expressing human PrP with new, robust phenotypes in brain neurons and the eye. Using comparable attP2 insertions, we demonstrate the heightened toxicity of human PrP compared to rodent PrP along with a specific interaction with the amyloid-β peptide. Using this new model, we started to uncover the intrinsic (sequence / structure) and extrinsic (interactions) factors regulating PrP toxicity. We describe PERK and ATF4as key cellular mechanism mediating the toxicity of human PrP and uncover a key new protective activity for 4E-BP, an ATF4 transcriptional target. Lastly, mutations in human PrP (N159D, D167S, N174S) show partial protective activity, revealing its high propensity to misfold into toxic conformations.

Intrinsic determinants of prion protein neurotoxicity in Drosophila: from sequence to (dys)function

“…In contrast, rabbit PrP-S174N had no effect on flies. Unexpectedly, expression of human PrP carrying the reciprocal substitutions (N159D, D167S, and N174S) showed little protective effect in fly toxicity, if any, indicating that these residues do not confer conformational stability of human PrP on their own ( Myers et al, 2022 , 2023 ). Considering the inconsistent effects of these substitutions and their dependence on the PrP backbone, the role of D159, S167, and S174 in the dynamics of the CT3DD is unclear.…”

“…The first fly models of prionopathy established that flies replicate key features of prion diseases, including age-dependent neurodegeneration and misfolding into disease-relevant conformations ( Gavin et al, 2006 ; Fernandez-Funez et al, 2009 ). We and others followed by showing that Drosophila expressing PrP-WT from dog, horse, or rabbit display no neurotoxicity and lower aggregation compared to those expressing mouse, hamster, ovine, bovine, or human PrP ( Fernandez-Funez et al, 2009 , 2010 ; Thackray et al, 2012b , 2021 ; Sanchez-Garcia and Fernandez-Funez, 2018 ; Myers et al, 2022 ). The preservation of intrinsic properties, including biogenesis, folding, and age-dependent misfolding and toxicity, demonstrates that flies provide an appropriate cellular environment for expressing mammalian PrP.…”

“…We generated transgenic models carrying human and rodent PrP constructs and showed that human PrP is qualitatively more toxic than mouse or hamster PrP ( Bischof et al, 2007 ). Human PrP is so far the only PrP model with a robust eye phenotype ( Fernandez-Funez et al, 2017 ; Myers et al, 2022 ), which is critical for performing fast phenotypic screens and has played a critical role in many Drosophila models of neurodegeneration ( Jackson et al, 1998 ; Warrick et al, 1998 ; Fernandez-Funez et al, 2000 ; Kazemi-Esfarjani and Benzer, 2000 ; Wittmann et al, 2001 ; Jackson et al, 2002 ; Crowther et al, 2005 ; Ritson et al, 2010 ; Casas-Tinto et al, 2011 ; Mizielinska et al, 2014 ). It is noteworthy that human PrP-WT is highly toxic when expressed in fly neurons.…”

“…The fast misfolding and toxicity in flies can be explained by a shift in conformational dynamics due to relative high expression and the hijacking of the biogenies and secretion cellular machinery due to the lack of endogenous PrP. It is important to remember that high expression of PrP from rabbit, dog, or horse PrP are not toxic in flies, whereas rodent PrP cause weaker phenotypes than human PrP ( Fernandez-Funez et al, 2010 ; Sanchez-Garcia and Fernandez-Funez, 2018 ; Myers et al, 2022 ). Thus, the high toxicity of human PrP must be encoded in its subtle sequence differences with animal PrPs.…”

“…Once the constructs are introduced in flies, a first generation cross can tell us in 10 days whether a mutation has any impact on the eye in living flies under the stereoscope. We recently introduced several candidate residues expected to lower PrP toxicity with unequal results ( Myers et al, 2022 , 2023 ). We examined the functional impact of introducing N/D159, D/S167, and N/S174 in the context of dog, horse, rabbit, mouse, or human PrP in transgenic flies.…”