BackgroundZebrafish Models of Alzheimer & Prion Disease. We are creating transgenic and mutant zebrafish toward the goal of modeling disease progression. We seek tractable disease models, or at least in vivo assays of protein function, to enable screening of candidate genes or small molecules as putative therapeutics. We have a goal of creating prion-infectible fish. A second approach is to explore genetic interactions and familial disease mutations. To complete these objectives we have improved upon and successfully deployed zinc finger nucleases, TALENS and CRISPR allowing targeted knockout of genes-of-interest. We also have created a variety of topical transgenic fish. We will continue to assess how these genetic manipulations affect how disease spreads through the CNS and affect synaptogenesis, including through collaborative in vivo electrophysiology approaches.
PrP as APP interactor.
The APP holoprotein is cleaved to give rise to Aβ, the toxic protein that aggregates into oligomers and plaques associated with neuron death in Alzheimer Disease. It is now accepted that PrPC is a receptor for Aβ; We have expanded this research space by showing that PrPC is also an interactor with the APP holoprotein. The interaction occurs at the level of biochemistry and synergistic toxic knockdown when APP and PrPC are knocked down in zebrafish. Further investigations will dissect this interaction.
Prion-like diseases and loss-of-function.
We assert that loss of protein function during misfolding, e.g. for APP and PrPC
, contributes substantially to the etiology of neurodegeneration. Disruption of APP or PrPC
levels/functions are promising therapeutic routes to treat Alzheimer disease. Thus we seek to fill a vast knowledge gap regarding the normal roles of APP and PrPC
as they pertain to Alzheimer Disease. Zebrafish provide a compelling platform for such studies because knockout of these proteins reveals highly tractable and relevant phenotypes (e.g. seizure susceptibility, excitotoxicity, sleep disorders) while their genetic pliability enables dissection of the molecular components behind the phenotypes.
Conserved functions of PrPC. We have
demonstrated that mammalian PrPC can rescue phenotypes in knockout zebrafish, demonstrating deeply conserved (important!) functions in cell adhesion and modulation of neural excitability. We will characterize purposefully engineered APP and PrP mutant zebrafish in vivo via a cutting-edge fluorescence reporter system that quantifies neural activity; this will test PrPC and APP requirements for seizures, hyperexcitability, and excitotoxicity, and enable mapping of which neurons and brain centres mediate such dysfunction with unprecedented specificity.
We will deploy our engineered zebrafish into high-throughput drug screening platforms. The zebrafish allows compelling in vivo drug screens that permit unprecedented detection of lead compounds (that would be missed in simpler systems) because the immense complexity of CNS cell physiology is well represented and because metabolism of drugs by diverse differentiated cell types is often required for the drug’s action.
Leighton PLA, WT Allison*. 2016. Protein misfolding in prion and prion-like diseases: reconsidering a required role for protein loss of function. J Alzheimer Disease. 54:3-29. DOI: 10.3233/JAD-160361
Huc-Brandt S, N Hieu, T Imberdis, N Cubedo, M Silhol, PLA Leighton, T Domachke, WT Allison, V Perrier, M Rossel. 2014. Zebrafish prion PrP2 controls collective migration process during lateral line sensory system development. PLoS ONE 9: e113331. doi:10.1371/journal.pone.0113331
Fleisch VC1, PLA Leighton1, H Wang, LM Pillay, RG Ritzel, G Bhinder, B Roy, KB Tierney, DW Ali, AJ Waskiewicz, WT Allison*. 2013. Targeted mutation of the gene encoding prion protein in zebrafish reveals a conserved role in neuron excitability. Neurobiology of Disease. 55:11-25. Cover Photo. doi:10.1016/j.nbd.2013.03.007 1equal contributors.
Kaiser DM1, M Acharya1, PLA Leighton1, H Wang, N Daude, S Wohgelmuth, B Shi, WT Allison*. 2012. Amyloid β Precursor Protein and Prion Protein have a conserved interaction affecting cell adhesion and CNS development. PLoS ONE. 7(12):e51305. 1equal contributors. doi:10.1371/journal.pone.0051305