UC San Diego

Prion diseases are fatal neurodegenerative disorders characterized by neuronal loss and the accumulation of misfolded prion protein aggregates (PrPSc) in the brain. PrPC, the normal cellular prion protein encoded by the Prnp gene, has been implicated in binding oligomers, including PrPSc, Aꞵ oligomers, and ⍺-synuclein assemblies, that form in patient brains with neurodegenerative disease. However, the mechanism by which PrPC interacts with PrPSc and causes neuronal death is poorly defined. The Prnp92N point mutation may be a tool to help to elucidate the neurotoxic pathway, as it has been shown to result in spontaneous spongiform degeneration and necrosis of hippocampal pyramidal neurons in mice despite the absence of PrPSc and prion infectivity. Autophagy may be one cellular mechanism affected by this mutation, as Prnp92N mice exhibit increased levels of p62 in whole brain lysate and autophagosome accumulation 20 days after birth, consistent with dysregulated autophagy in other neurodegenerative disease models. Western blot results from Prnp92N cerebral cortex show a strong increase in pULK-S757/ULK1 levels at 10 days post-birth. There was also increased p62, a protein degraded by autophagy, and LC3B, an autophagosome marker, consistent with a reduction in autophagic flux. Inhibition of autophagic machinery has previously been shown in prion disease, and understanding how autophagic function changes in Prnp92N mice may help to uncover the function of PrPC in prion disease and point towards potential therapeutic avenues.

Additional models are needed to validate in vivo results and better substantiate the role of PrPC in biological systems. Due to their low cost, rapid reproduction rate, short life span, and susceptibility to genetic manipulation, Drosophila melanogaster are an excellent candidate for studying prion-induced neurodegeneration. To investigate prion disease in a non-mammalian model, we generated transgenic UAS-Prnp92N and wild type mouse (WT) PrP control Drosophila lines, which were sequence validated by PCR. Western blot analysis revealed that the UAS-Prnp92N point mutation is uniquely expressed in the UAS-Prnp92N fly lines, and that overall PrPC levels in UAS-Prnp92N and WT flies are similar. Lifetime and climbing assays show a reduction in lifespan and motor ability in UAS-Prnp92N cohorts, consistent with other prion disease models in Drosophila. These experiments show the generation of a mutant prion protein disease model in Drosophila, and future experiments will focus on assessing behavior, histopathology, and signaling pathways towards a goal of understanding how mutant prion protein impacts autophagy and induces neuronal death.